Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to compounds that modulate BRAF activity.
• the invention relates in particular to a compound of formula (I)
• the Rapidly Accelerated Fibrosarcoma (RAF) class of serine-threonine kinases comprise three members (ARAF, BRAF, RAF1) that compose the first node of the MAP kinase signalling pathway.
• ARAF Rapidly Accelerated Fibrosarcoma
• Mutant BRAF is a targetable oncogenic driver and three BRAF inhibitors (vemurafenib, dabrafenib and encorafenib) reached the market up to now showing efficacy in BRAFV600E-positive melanoma.
• BRAFV600E-positive melanoma the number of BRAF inhibitors that are currently being explored.
• rapid acquisition of drug resistance is almost universally observed and the duration of the therapeutic benefits for the targeted therapy remains limited.
• BRAF inhibitors revealed an unexpected and “paradoxical” ability to repress MAPK signalling in BRAFV600E-driven tumours while the same inhibitors presented MAPK stimulatory activities in BRAF wild type (WT) models (N Engl J Med 2012; 366:271-273; and British Journal of Cancer volume 111, pages 640-645(2014)).
• inhibitors like vemurafenib, dabrafenib or encorafenib to a WT BRAF or RAF1 protomer quickly induces RAF homo and/or hetero dimerization and membrane association of the newly formed RAF dimer.
• one RAF protomer allosterically induces conformational changes of the second resulting in a kinase active status and, importantly, in a conformation unfavourable for the binding of the inhibitor.
• the dimer induced by drug treatment promotes MEK phosphorylation by the catalysis operated by the unbound protomer with hyperactivation of the pathway.
• the RAF paradox results in two clinically relevant consequences: 1) accelerated growth of secondary tumours upon BRAFi monotherapy (mainly keratochantoma and squamous-cell carcinomas) (N Engl J Med 2012; 366:271-273) and 2) the acquisition of drug resistance in the setting of BRAFi monotherapy as well as in combinations of BRAFi+MEKi presents activation of dimer-mediated RAF signalling by genetically driven events including RAS mutations, BRAF amplifications, expression of dimeric-acting BRAF splice variants (Nature Reviews Cancer volume 14, pages 455-467(2014)).
• the present invention relates to the surprising finding that the BRAF inhibitor of formula (I) shows considerably less paradoxial activation of the MAPK signalling pathway while retaining high potency.
• This compound can also be referred to as a paradox breaker or RAF paradox breaker, compared to compounds inducing the RAF paradox (and which could be referred to as paradox inducers or RAF paradox inducers).
• alkyl signifies a straight-chain or branched-chain alkyl group with 1 to 8 carbon atoms, particularly a straight or branched-chain alkyl group with 1 to 6 carbon atoms and more particularly a straight or branched-chain alkyl group with 1 to 4 carbon atoms.
• Examples of straight-chain and branched-chain C 1 -C 8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert ⁇ -butyl, the isomeric pentyls, the isomeric hexyls, the isomeric heptyls and the isomeric octyls, particularly methyl, ethyl, propyl, butyl, pentyl and hexyl.
• Methyl, ethyl, propyl and pentyl are particular examples of “alkyl” in the compound of formula (I).
• cycloalkyl signifies a cycloalkyl ring with 3 to 8 carbon atoms and particularly a cycloalkyl ring with 3 to 6 carbon atoms.
• Examples of “cycloalkyl” are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctanyl.
• Particular examples of “cycloalkyl” are cyclopropyl, cyclobutyl and cyclopentyl.
• alkoxy or “alkyloxy”, alone or in combination, signifies a group of the formula alkyl-O— in which the term “alkyl” has the previously given significance, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert ⁇ -butoxy.
• alkoxy are methoxy and ethoxy.
• halogen or “halo”, alone or in combination, signifies fluorine, chlorine, bromine or iodine and particularly fluorine, chlorine or bromine, more particularly fluorine.
• halo in combination with another group, denotes the substitution of said group with at least one halogen, particularly substituted with one to five halogens, particularly one to four halogens, i.e. one, two, three or four halogens.
• alkylamino alone or in combination, signifies an alkyl group linked to a —NH-group.
• dialkylamino alone or in combination, signifies two alkyl groups linked to a —N-atom.
• carbonyl alone or in combination, signifies the —C(O)— group.
• amino alone or in combination, signifies the primary amino group (—NH 2 ), the secondary amino group (—NH—), or the tertiary amino group (—N—).
• cyano alone or in combination, signifies the —CN group.
• heterocycloalkyl denotes a monovalent saturated or partly unsaturated mono- or bicyclic ring system of 4 to 9 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon. Bicyclic means consisting of two cycles having one or two ring atoms in common.
• heterocycloalkyl examples include pyrrolidinyl, piperidinyl, 1,3-dihydroisoindolyl, 1H-isoquinolinyl, azetidinyl, and piperazinyl.
• Particular examples of “heterocycloalkyl” are pyrrolidinyl, piperidinyl and piperazinyl.
• salts refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable.
• the salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein.
• salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts.
• Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.
• the compound of formula (I) can also be present in the form of zwitterions.
• Particularly preferred pharmaceutically acceptable salts of the compound of formula (I) are the salts of trifluoroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and methanesulfonic acid.
• one of the starting materials or compounds of formula (I) contain one or more functional groups which are not stable or are reactive under the reaction conditions of one or more reaction steps
• appropriate protecting groups as described e.g. in “Protective Groups in Organic Chemistry” by T. W. Greene and P. G. M. Wuts, 3 rd Ed., 1999, Wiley, New York
• Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature.
• protecting groups are tert-butoxycarbonyl (Boc), 9-fluorenylmethyl carbamate (Fmoc), 2-trimethylsilylethyl carbamate (Teoc), carbobenzyloxy (Cbz) and p-methoxybenzyloxycarbonyl (Moz).
• the compound of formula (I) can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
• asymmetric carbon atom means a carbon atom with four different substituents. According to the Cahn-Ingold-Prelog Convention an asymmetric carbon atom can be of the “R” or “S” configuration.
• the invention thus relates to:
• the invention further relates to a compound of formula (I) selected from
• the invention further relates to a compound of formula (I) selected from
• the preparation of compounds of formula (I) of the present invention may be carried out in sequential or convergent synthetic routes. Syntheses of the invention are shown in the following general schemes. The skills required for carrying out the reactions and purifications of the resulting products are known to those persons familiar with the art. In case a mixture of enantiomers or diastereoisomers is produced during a reaction, these enantiomers or diastereoisomers can be separated by methods described herein or known to those skilled in the art such as e.g. (chiral) chromatography or crystallization. The substituents and indices used in the following description of the processes have the significance given herein.
• ester B can be reduced to alcohol C with a variety of reducing agents such as LiAlH 4 , DIBAL, SMEAH (Red-Al) or the like in solvents such as THF, ether, DCM or similar at temperatures ranging from ⁇ 78 to the boiling point of the solvent.
• Oxidation to aldehyde D can then be achieved by treatment with a variety of oxidizing agent such as MnO 2 , Dess-Martin periodinane, pyridinium chlorochromate (PCC) or similar, in an appropriate solvent such as DCM, CHCl 3 , THF, ether or the like at various temperatures.
• aldehyde D might be accessible by direct reduction of ester B with appropriate reducing agents such as DIBAL in an appropriate solvent such as THF, DCM or the like at low temperatures ranging from ⁇ 78° C. to 0° C.
• an aldehyde D and aryl-acetic acid esters of formula E can be treated with a base such as potassium carbonate, cesium carbonate or the like in a solvent such as DMF, THF, CH 3 CN or the like, at temperatures ranging from rt to the boiling point of the solvent.
• intermediate F can be treated with a sulfonylchloride or sulfamoylchloride of formula L, where R 1 is either (branched) lower alkyl or a secondary amine substituted by lower alkyl/cycloalkyl, in the presence of a base such as NEt 3 , DIPEA, K 2 CO 3 , Cs 2 CO 3 or the like in solvents such as DCM, THF, CHCl 3 DMF, CH 3 CN or similar or in pyridine optionally with or without DMAP.
• a base such as NEt 3 , DIPEA, K 2 CO 3 , Cs 2 CO 3 or the like
• solvents such as DCM, THF, CHCl 3 DMF, CH 3 CN or similar or in pyridine optionally with or without DMAP.
• an ester of formula H can be used as a starting material and similar reactions conditions as described for the transformation of E to F can be applied.
• the methylsulfonyl group can be displaced by treatment of either L or N with the appropriate amine R 5 -L-NH 2 in a solvent such as ether, dioxane, THF, DCM, DMF CH 3 CN or the like at various temperatures ranging from ⁇ 20° C. to the boiling point of the solvent.
• a base such as NEt 3 , DIPEA, K 2 CO 3 , Cs 2 CO 3 or similar can be present in this reaction, which will provide the compounds of formula M or O.
• introduction of the desired sulfonylamino- or sulfamoylamino group to provide intermediates or examples of formula Q can be achieved by applying for example a Buchwald type protocol in the presence of sulfonamid or sulfamoylamide P and a suitable catalyst such as Pd 2 (dba) 3 and a ligand such as 2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl in an appropriate solvent such as dioxane, DMF, toluene or similar, optionally in the presence of a base such as NaO tert Bu, Cs 2 CO 3 , K 2 CO 3 , Na 2 CO 3 or similar at temperatures ranging from rt to the boiling point of the solvent either with or without microwave irradiation.
• a base such as NaO tert Bu, Cs 2 CO 3 , K 2 CO 3 , Na 2 CO 3 or similar at temperatures ranging from rt to
• an intermediate or example carries a side chain R 4 or R 5 of the structure type R shown in scheme 3 with protected primary amine
• treatment with an acid HXc such as HCl, HBr, TFA or the like in a solvent such as water, dioxane, DCM, CHCl 3 or similar will provide intermediates or examples of formula S with a free amine side chain.
• the side chain can be in a salt form as shown in scheme 3, structure S, or alternatively as a free amine (not shown).
• intermediates or examples with a side chain R 4 or R 5 represented by structure S can be treated with an activated carboxylic acid T, where Ra is for example lower alkyl and LG is a leaving group and represents an activated carboxylic acid such as a carboxylic acid halide, anhydride, imidazolide or an intermediate coming from activation of the carboxylic acid with a peptide coupling reagent such as for example HATU, optionally in the presence of a base such as NEt 3 , DIPEA, NaHCO 3 , Na 2 CO 3 or similar, in solvents such as water, DCM, THF, DMF or the like depending on the reagents used, to provide examples of formula U with R 4 or R 5 carrying an acyl-amino group.
• a peptide coupling reagent such as for example HATU
• an intermediate or example carries a side chain R 5 of the structure type Y shown in scheme 4 with an ester group COORc
• Rb is tert Bu
• treatment with an acid HXd such as HCl, HBr, TFA or the like in a solvent such as dioxane, DCM, CHCl 3 or similar will provide intermediates or examples of formula Z with a free carboxylic acid side chain.
• Rb is for example methyl or ethyl or similar
• a base such as NEt 3 , DIPEA, NaHCO 3 , Na 2 CO 3 or the like
• R 5 incorporates a phenyl group substituted by cyano as shown in scheme 5
• structure AE can be reduced to provide an aminomethyl derivative of formula AF.
• Conditions for this step are for example treatment with a reducing agent such as NaBH 4 or LiBH 4 , optionally in the presence CoCl 2 or similar, or other reducing agents such as DIBAH.
• a reducing agent such as NaBH 4 or LiBH 4
• CoCl 2 or similar or other reducing agents
• DIBAH reducing agents
• amine intermediate AF can be optionally acylated with reagents of formula T to provide acyl derivatives of formula AG.
• the starting material for the introduction of the desired side chains R 5 in form of the amines R 5 -L-NH 2 needed to be synthesized.
• the synthesis of such starting materials is outlined in scheme 6 below.
• an appropriately substituted phenylacetic acid ester AH can be treated with a suitably protected aminoethyltrifluoroborate reagent AI, where PG represents for example a compatible benzyloxycarbonyl group or similar, to provide intermediate AJ.
• the transformation is carried out in the presence of an appropriate Pd catalyst such as 1.1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride DCM complex or similar, in the presence of abase such as K 2 CO 3 , Cs 2 CO 3 or KO tert Bu or the like in a solvent such as water/toluene, water/dioxane, DMF or the like at temperatures ranging from rt to the boiling point of the solvent either with or without microwave irradiation.
• an appropriate Pd catalyst such as 1.1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride DCM complex or similar
• abase such as K 2 CO 3 , Cs 2 CO 3 or KO tert Bu or the like
• a solvent such as water/toluene, water/dioxane, DMF or the like at temperatures ranging from rt to the boiling point of the solvent
• Deprotection of AJ to give amine AK can be achieved by catalytic hydrogenation with a suitable catalyst such as Pd/C or Pd(OH) 2 or similar in an appropriate solvent such as MeOH, EtOH or EtOAc, if PG is a benzyloxycarbonyl protecting group.
• a suitable catalyst such as Pd/C or Pd(OH) 2 or similar in an appropriate solvent such as MeOH, EtOH or EtOAc, if PG is a benzyloxycarbonyl protecting group.
• suitably protected cyclic amine precursors AN where PG* is an orthogonal protecting group as compared to PG and n is e.g. 1 or 2, can be made in an analogous way.
• PG* is an orthogonal protecting group as compared to PG and n is e.g. 1 or 2
• n is e.g. 1 or 2
• catalysts such as for example methanesulfonato(2-dicyclohexylphosphino-2′-6′-di-i-propoxy-1,1′-biphenyl)(2-amino-1-1′biphenyl-2-yl)palladium(II) may be more appropriate for this transformation.
• the transformation from AM to AN can be carried out as describe above for AJ to AK.
• an appropriate arylacetic acid ester intermediate H where R 3 is an electron withdrawing group such as CN, needed to be prepared.
• a possible approach is outlined in scheme 7.
• a commercially available, suitably substituted ortho-F-benzonitrile AO can be treated for example with diethylmalonate or similar in the presence of a base such as NaH, KO tert Bu, LDA or the like in a solvent such as THF, ether or dioxane or similar at temperatures from ⁇ 20° C. to the boiling point of the solvent to afford intermediate AP.
• AP can be decarboxylated for example by treatment with LiCl in DMSO at elevated temperatures from 40-190° C. to provide arylacetic acid esters AQ.
• the invention thus also relates to a process for the preparation of a compound according to the invention, comprising one of the following steps:
• step (a) can conveniently be carried out in a solvent.
• the solvent can be for example DMF, THF, or a mixture thereof.
• the base can be for example triethylamine or potassium carbonate.
• the base is triethylamine.
• Convenient conditions for the reaction of step (a) can be between around 0° C. and around 50° C., particularly between around 5° C. and around 30° C., more particularly between around 10° C. and around 30° C.
• Preferred conditions for the reaction of step (a) are the use of triethylamine in a DMF at around room temperature for between around 1-24 hrs, in particular between around 5-15 hrs.
• the reaction of step (b) can conveniently be carried out in a solvent.
• the solvent can be for example dioxane, THF, or a mixture thereof.
• the base can be for example cesium carbonate or potassium carbonate. Conveniently the base is cesium carbonate.
• Convenient conditions for the reaction of step (b) can be between around 50° C. and around 100° C., particularly between around 60° C. and around 100° C., more particularly between around 70° C. and around 80° C.
• Preferred conditions for the reaction of step (b) are the use of cesium carbonate in dioxane at around 80° C. for between around 10-24 hrs, in particular between around 15-18 hrs.
• the invention also relates to a compound according to the invention when manufactured according to a process of the invention.
• the compound of formula (I) may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
• physiologically acceptable carriers i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
• the pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8.
• a compound of formula (I) is formulated in an acetate buffer, at pH 5.
• the compound of formula (I) is sterile.
• the compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.
• compositions are formulated, dosed, and administered in a fashion consistent with good medical practice.
• Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
• the compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration.
• Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
• the compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc.
• Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
• a typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient.
• Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients.
• the formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
• buffers stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing
• the invention also relates in particular to:
• a certain embodiment of the invention relates to the compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, for the use in the treatment or prophylaxis of cancer, in particular melanoma or NSCLC, characterized by a BRAF mutation selected from V600E and V600K.
• a certain embodiment of the invention relates to a method for the treatment or prophylaxis of cancer, in particular melanoma or NSCLC, wherein a BRAF mutation selected from V600E and V600K is present in the cancer, which method comprises administering an effective amount of a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, to a patient in need thereof.
• the invention includes all substituents in its corresponding deuterated form, wherever applicable, of the compound of formula (I).
• the invention includes the corresponding carboxylic acid of the ester form, wherever applicable, of the compound of formula (I).
• the invention includes all optical isomers, i.e. diastereoisomers, diastereomeric mixtures, racemic mixtures, all their corresponding enantiomers and/or tautomers as well as their solvates, wherever applicable, of the compound of formula (I).
• Step 1 (3R)—N-[2,4-difluoro-3-(8-methyl-2-methylsulfanyl-7-oxopyrido[2,3-d]pyrimidin-6-yl)phenyl]-3-fluoropyrrolidine-1-sulfonamide
• Step 2 (3R)—N-[2,4-difluoro-3-(8-methyl-2-methylsulfonyl-7-oxopyrido[2,3-d]pyrimidin-6-yl)phenyl]-3-fluoropyrrolidine-1-sulfonamide
• the reaction mixture was diluted with 40 ml DCM and 40 ml sat NaHCO 3 and the layers were separated. The aqueous layer was extracted with 3 ⁇ 40 ml DCM. The organic extracts were washed with 40 ml water, dried with anhydrous sodium sulfate, filtered and concentrated in vacuo to yield 1.52 g of a crude product.
• This material was dissolved in DCM/MeOH and was adsorbed to silica gel. The dried silica gel was applied to a precolumn fitted in front of a 80 g silica gel column. The column was eluted with a gradient of 0-70% ethyl acetate in heptane. Fractions containing pure product were combined and concentrated to give 1.15 g (81%) of the desired material as a pale yellow foam. MS (ESI): 518.2 [MH + ].
• Step 1 4-[[6-[2,6-difluoro-3-[[(3R)-3-fluoropyrrolidin-1-yl]sulfonylamino]phenyl]-8-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]butanoic acid
• Step 2 tert-butyl 4-[4-[[6-[2,6-difluoro-3-[[(3R)-3-fluoropyrrolidin-1-yl]sulfonylamino]phenyl]-8-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]butanoylamino]piperidine-1-carboxylate
• Step 1 tert-Butyl N-methyl-N-[[3-[2-(phenylmethoxycarbonylamino)ethyl]phenyl]methyl]carbamate
• Step 2 tert-butyl N-[[3-(2-aminoethyl)phenyl]methyl]-N-methylcarbamate
• Step 3 tert-Butyl N-[[3-[2-[[6-[2,6-difluoro-3-[[(3R)-3-fluoropyrrolidin-1-yl]sulfonylamino]phenyl]-8-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]ethyl]phenyl]methyl]-N-methylcarbamate
• the reaction was cooled to rt and most of the THF was removed in vacuo.
• the residue was diluted with 1 M aq. HCl (150 mL) and was extracted with TBME (3 ⁇ 150 mL).
• the combined organic extracts were washed with water (300 mL) and brine (300 mL), dried (MgSO 4 ), filtered and concentrated in vacuo to provide 5.12 g of a yellow oil that contained the diester intermediate.
• Step 4 tert-Butyl 4-[2-[[6-(3-bromo-2-cyanophenyl)-8-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]ethyl]piperidine-1-carboxylate
• Step 5 tert-Butyl 4-[2-[[6-[2-cyano-3-[[ethyl(methyl)sulfamoyl]amino]-phenyl]-8-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]ethyl]piperidine-1-carboxylate
• Step 1 Ethyl 4-[2-[2-[(2-methylpropan-2-yl)oxycarbonylamino]ethoxy]ethoxy]ethyl amino]-2-methylsulfanylpyrimidine-5-carboxylate
• Step 2 tert-Butyl N-[2-[2-[[5-(hydroxymethyl)-2-methylsulfanylpyrimidin-4-yl]amino]ethoxy]ethoxy]ethyl]carbamate
• Step 3 tert-Butyl N-[2-[2-[(5-formyl-2-methylsulfanylpyrimidin-4-yl)amino]ethoxy]ethoxy]ethyl]carbamate
• Step 4 tert-Butyl N-[2-[2-[6-(3-amino-2,6-difluorophenyl)-2-methylsulfanyl-7-oxopyrido[2,3-d]pyrimidin-8-yl]ethoxy]ethoxy]ethyl]carbamate
• the reaction mixture was then concentrated in vacuo to remove most of the DMF and was then diluted with 40 ml ethyl acetate and 40 ml 2M Na 2 CO 3 and the layers were separated.
• the aqueous layer was extracted with 2 ⁇ 30 ml ethyl acetate and the combined organic layers washed with 30 ml brine.
• the organic extract was dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuo and the remainder of the DMF was removed by co-evaporation with 2 ⁇ 50 ml toluene. This afforded 1.16 g of a brown oil.
• Step 5 tert-Butyl N-[2-[2-[6-[2,6-difluoro-3-[[(3R)-3-fluoropyrrolidin-1-yl]sulfonylamino]phenyl]-2-methylsulfanyl-7-oxopyrido[2,3-d]pyrimidin-8-yl]ethoxy]ethoxy]ethyl]carbamate
• Step 6 tert-Butyl N-[2-[2-[6-[2,6-difluoro-3-[[(3R)-3-fluoropyrrolidin-1-yl]sulfonylamino]phenyl]-2-methylsulfonyl-7-oxopyrido[2,3-d]pyrimidin-8-yl]ethoxy]ethoxy]ethyl]carbamate
• Step 7 tert-Butyl N-[2-[2-[2-[2-(cyclopropylmethylamino)-6-[2,6-difluoro-3-[[(3R)-3-fluoropyrrolidin-1-yl]sulfonylamino]phenyl]-7-oxopyrido[2,3-d]pyrimidin-8-yl]ethoxy]ethoxy]ethyl]carbamate
• Step 1 tert-Butyl 5-[2-(phenylmethoxycarbonylamino)ethyl]-1,3-dihydroisoindole-2-carboxylate
• Step 2 tert-Butyl 5-(2-aminoethyl)-1,3-dihydroisoindole-2-carboxylate
• Step 1 (3R)—N-[3-[2-[2-[3-(aminomethyl)phenyl]ethylamino]-8-methyl-7-oxopyrido[2,3-d]pyrimidin-6-yl]-2,4-difluorophenyl]-3-fluoropyrrolidine-1-sulfonamide
• Step 2 N-[[3-[2-[[6-[2,6-difluoro-3-[[(3R)-3-fluoropyrrolidin-1-yl]sulfonylamino]phenyl]-8-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]ethyl]phenyl]methyl]acetamide
• the reaction mixture was diluted with 10 ml DCM and 10 ml water. The layers were separated and the aqueous layer extracted with additional 2 ⁇ 10 ml DCM. The combined organic layers were washed with 10 ml brine, dried with anhydrous sodium sulfate, filtered and concentrated in vacuo to give 4.9 mg of a crude product.
• A375 is a cellular cancer model expressing V600E mutated BRAF and HCT116 a cellular cancer model expressing WT BRAF.
• First generation BRAF inhibitors such as e.g. dabrafenib induce a paradox effect on tumour cells in that they inhibit the growth of V600E mutated BRAF cells (such as e.g. A375), while they activate growth in WT BRAF cells (such as e.g. HCT 116).
• ERK 1,2 phosphorylation (terminal member of the phosphorylation cascade of the MAPK pathway) is hereafter reported as main readout for the activation status of the MAPK pathway.
• DMEM no-phenol red medium supplemented with L-glutamine was purchased from (Thermo Fisher Scientific).
• Fetal bovine serum was purchased from VWR. Advanced ERK phospho-T202/Y204 kit—10,000 tests was purchased from Cisbio cat #64AERPEH. A375 (catalog #CRL-1619) and HCT116 (catalog #CCL-247) cells were originally obtained from American type Culture Collection (ATCC) and banked by the Roche repository. 384-Well microplates ultized were purchased from Greiner Bio-One, 384-well, (With Lid, HiBase, Low volume cat 784-080).
• A375 and HCT116 cell lines are maintained in DMEM no-phenol red medium supplemented with 10% fetal bovine serum (FBS).
• FBS fetal bovine serum
• P-ERK levels are determined by measuring FRET fluorescence signal induced by selective binding of two antibodies provided in the mentioned kit (Cisbio cat #64AERPEH) on ERK protein when phosphorylated at Thr202/Tyr204. Briefly, 8000 cells/well in 12 ⁇ l media/well are plated in the 384-well plate and left overnight in the incubator (at 37° C.
• the plate is treated in duplicate with test compounds, dabrafenib and PLX8394 (the latter two as controls) at the following final drug concentrations: 10 ⁇ M, 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M, 0.1 ⁇ M, 0.03 ⁇ M, 0.01 ⁇ M, 0.003 ⁇ M and 0.001 ⁇ M. All wells are subjected to DMSO normalization and drug incubation occurs for 1 hour. Then, 4 ⁇ l of a 4 ⁇ lysis buffer supplied with the kit are added to the wells, the plate is then centrifuged for 30 second (3000 rpm) and incubated on a plate shaker for 1 h at rt.
• p-ERK HTRF well compositions ( ⁇ l): neg ctrl pos ctrl neut ctrl cpd blank — — 12 12 12 Cells 12 — — — — Media — — — ⁇ 0.05 — Cpd — 16 — — — control lysate (ready-to- use) 4 — 4 4 4 4x lysis buffer 4 4 4 4 — Advanced p-ERK antibody solution — — — — 4 Advanced p-ERK1/2 Cryptate antibody solut. 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
• the plate is then centrifuged at 3000 rpm for 30 seconds, sealed to prevent evaporation and incubated overnight in the dark at room temperature.
• the plate is then analyzed and fluorescence emission value collected through a Pherastast FSX (BMG Labtech) apparatus at 665 and 620 nM.
• Data are normalized in the case of A375 cells (BRAF inhibition) considering the average of the ratio (blank subtracted) derived by DMSO only treated cells as 100% and by considering the average of the ratio (blank subtracted) derived by 10 ⁇ M Dabrafenib treated cells as 0%. Mean of the normalized points are fitted with sigmoidal curve and IC 50 values are determined.
• Film coated tablets containing the following ingredients can be manufactured in a conventional manner:
• Kernel Compound of formula (I) 10.0 mg 200.0 mg Microcrystalline cellulose 23.5 mg 43.5 mg Lactose hydrous 60.0 mg 70.0 mg Povidone K30 12.5 mg 15.0 mg Sodium starch glycolate 12.5 mg 17.0 mg Magnesium stearate 1.5 mg 4.5 mg (Kernel Weight) 120.0 mg 350.0 mg Film Coat: Hydroxypropyl methyl cellulose 3.5 mg 7.0 mg Polyethylene glycol 6000 0.8 mg 1.6 mg Talc 1.3 mg 2.6 mg Iron oxide (yellow) 0.8 mg 1.6 mg Titan dioxide 0.8 mg 1.6 mg
• the active ingredient is sieved and mixed with microcrystalline cellulose and the mixture is granulated with a solution of polyvinylpyrrolidone in water. The granulate is then mixed with sodium starch glycolate and magnesium stearate and compressed to yield kernels of 120 or 350 mg respectively. The kernels are lacquered with an aq. solution/suspension of the above mentioned film coat.
• Capsules containing the following ingredients can be manufactured in a conventional manner:
• the components are sieved and mixed and filled into capsules of size 2.
• Injection solutions can have the following composition:
• the active ingredient is dissolved in a mixture of Polyethylene glycol 400 and water for injection (part).
• the pH is adjusted to 5.0 by addition of acetic acid.
• the volume is adjusted to 1.0 ml by addition of the residual amount of water.
• the solution is filtered, filled into vials using an appropriate overage and sterilized.

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